Leptin plays a critical role in regulating systemic energy balance. This hormone's role in signaling the status of peripheral fat stores to the central nervous system, and in regulating energy intake and expenditure are well documented. The goal of this proposal is to address two less-well defined, but critically related, aspects of the biology of leptin. Specific Aim I is to investigate the molecular mechanisms responsible for the close correlation between leptin gene expression, adipocyte size and the metabolic status of adipocytes. Specific Aim II is to investigate the long-term effects of direct leptin action on metabolism and gene expression in adipocytes, and the contribution of direct leptin action in adipocytes to systemic energy homeostasis. In Specific Aim I, the hypothesis being tested is that the metabolic status of adipocytes is a primary determinant of leptin gene expression, a unifying mechanism responsible for signaling adipocyte size in fed states (long term energy balance) and acute caloric deficit in fasting states (short-term energy balance). The metabolic status of adipocytes will be manipulated using nutritional, pharmacological and molecular genetic tools, and the effects of such manipulations on relationships of leptin gene expression with the metabolic status and size of adipocytes will be determined. Metabolic intermediates responsible for regulating leptin gene expression to reflect changes in adipocyte size and acute systemic caloric balance will be identified. In Specific Aim II, the hypothesis being tested is that direct leptin actions in adipocytes play a significant role in regulating local adipocyte metabolism and gene expression. Such local action of leptin may affect body fat distribution and depot-specific differences in adipocyte functions. A preadipocyte implantation system will be used to study direct leptin actions on local adipocyte functions in vivo. Adenovirus-mediated gene transfer will be used to modify leptin receptor function or the rate of leptin production in cultured preadipocytes, which will then be implanted in athymic nude mice. The effects of these modifications on adipocyte functions (metabolism and expression of secreted proteins) in the mature adipocytes derived from these implanted preadipocytes will be determined. The effects of direct leptin actions on the adipocyte functions in anatomically distinct fat depots will also be investigated in tissue-specific transgenic mouse models. A better understanding of the regulation of leptin production in adipocytes in response to changes in systemic energy balance will provide a basis for preventing the decrease of leptin production induced by dieting or weight loss, thus blocking a feedback regulatory mechanism that interferes with success in clinical weight loss efforts. The work on the role of direct leptin actions in regulating local adipocyte functions will provide insights into the mechanisms and molecules involved in the regulation of body fat distribution and development of depot-specific differences in adipocyte metabolism, and provide a basis for rationalizing the prevention and treatment of abdominal obesity and its metabolic co-morbidities, such as insulin resistance, dyslipidemia, hypertension, diabetes and cardiovascular diseases.